Antisense modulation of BCL2-associated X protein expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of BCL2-associated X protein. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding BCL2-associated X protein. Methods of using these compounds for modulation of BCL2-associated X protein expression and for treatment of diseases associated with expression of BCL2-associated X protein are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods formodulating the expression of BCL2-associated X protein. In particular,this invention relates to compounds, particularly oligonucleotides,specifically hybridizable with nucleic acids encoding BCL2-associated Xprotein. Such compounds have been shown to modulate the expression ofBCL2-associated X protein.

BACKGROUND OF THE INVENTION

[0002] Apoptosis, or programmed cell death, is a naturally occurringprocess that has been strongly conserved during evolution to preventuncontrolled cell proliferation. This form of cell suicide plays acrucial role in ensuring the development and maintenance ofmulticellular organisms by eliminating superfluous or unwanted cells.However, if this process becomes overstimulated, cell loss anddegenerative disorders including neurological disorders such asAlzheimers, Parkinsons, ALS, retinitis pigmentosa and blood celldisorders can result. Stimuli which can trigger apoptosis include growthfactors such as tumor necrosis factor (TNF), Fas and transforming growthfactor beta (TGFβ), neurotransmitters, growth factor withdrawal, loss ofextracellular matrix attachment and extreme fluctuations inintracellular calcium levels (Afford and Randhawa, Mol. Pathol., 2000,53, 55-63).

[0003] Alternatively, insufficient apoptosis, triggered by growthfactors, extracellular matrix changes, CD40 ligand, viral gene productsneutral amino acids, zinc, estrogen and androgens, can contribute to thedevelopment of cancer, autoimmune disorders and viral infections (Affordand Randhawa, Mol. Pathol., 2000, 53, 55-63). Consequently, apoptosis isregulated under normal circumstances by the interaction of gene productsthat either induce or inhibit cell death and several gene products whichmodulate the apoptotic process have now been identified.

[0004] Apoptosis manifests itself in two major downstream executionprograms: the caspase pathway and mitochondrial dysfunction (Korsmeyeret al., Cold Spring Harb. Symp. Quant. Biol., 1999, 64, 343-350;Korsmeyer et al., Cell Death Differ., 2000, 7, 1166-1173). The BCL-2family members play a pivotal role in determining whether a cell willlive or die by acting at the mitochondrial level. This family includesboth pro-apoptotic and anti-apoptotic proteins and the ratio betweenthese subsets is a determinant of the susceptibility of the cells todeath signals (Korsmeyer et al., Cold Spring Harb. Symp. Quant. Biol.,1999, 64, 343-350). Members of the BCL-2 family have the ability to formhomodimers or heterodimers, suggesting neutralizing competition amongthese proteins. An additional characteristic of functional significanceis the ability of BCL-2 family members to act as integral membraneproteins (Korsmeyer et al., Cold Spring Harb. Symp. Quant. Biol., 1999,64, 343-350).

[0005] BCL2-associated X protein (also known as BAX), is a pro-apoptoticBCL-2 family member. Activation of BCL2-associated X protein involvessubcellular translocation and dimerization. In viable cells, asubstantial portion of BCL2-associated X protein is monomeric and foundeither in the cytosol or loosely associated with membranes. Following adeath stimulus, cytosolic monomeric BCL2-associated X proteintranslocates to the mitochondria where it becomes a cross-linkable,integral membrane protein. The ability of BCL2-associated X protein toform distinct ion-conductive membrane pores may be, in part, responsiblefor mitochondrial dysfunction that leads to cell death (Korsmeyer etal., Cold Spring Harb. Symp. Quant. Biol., 1999, 64, 343-350; Korsmeyeret al., Cell Death Differ., 2000, 7, 1166-1173).

[0006] BCL2-associated X protein has been cloned (Oltvai et al., Cell,1993, 74, 609-619) and mapped to chromosome 19q13.3 (Apte et al.,Genomics, 1995, 26, 592-594; Chou et al., Cancer Genet. Cytogenet.,1996, 88, 136-140), and has been shown to encode a number of splicevariants, including BAX-alpha, BAX-beta, BAX-gamma (Oltvai et al., Cell,1993, 74, 609-619), BAX-delta (Apte et al., Genomics, 1995, 26,592-594), BAX-omega (Zhou et al., J. Biol. Chem., 1998, 273,11930-11936) and BAX-epsilon (Shi et al., Biochem. Biophys. Res.Commun., 1999, 254, 779-785.). Nucleotide sequences encoding BAX-alpha,BAX-beta and BAX-gamma are disclosed and claimed in U.S. Pat. Nos.5,691,179 and 5,955,595 (Korsmeyer, 1997; Korsmeyer, 1999). Nucleotidesequences encoding BAX-omega are disclosed and claimed in U.S. Pat. No.6,140,484 and corresponding PCT publication WO 97/01635 (Bitler et al.,2000; Bitler et al., 1997). Also disclosed in U.S. Pat. No. 6,140,484 isa 22-mer antisense oligonucleotide directed against the exon5/intron5junction of human BAX-omega (Bitler et al., 2000).

[0007] Overexpression of BCL2-associated X protein has been observed inhuman diseases including, glomerular disease (Yoshimura et al., Nephrol.Dial. Transplant, 1999, 14, 55-57), Hodgkin's disease (Brousset et al.,Blood, 1996, 87, 2470-2475), cartilage-hair hypoplasia (Yel et al., J.Clin. Immunol., 1999, 19, 428-434) and ocular complications arising fromdiabetes (Podesta et al., Am. J. Pathol., 2000, 156, 1025-1032). Inaddition, animal models of human disease have been used to determinethat overexpression of BCL2-associated X protein also may occur inAlzheimer's disease (MacGibbon et al., Brain Res., 1997, 750, 223-234),Parkinson's disease (Vila et al., Proc. Natl. Acad. Sci. U.S. A., 2001,98, 2837-2842), familial amyotrophic lateral sclerosis (Vukosavic etal., J. Neurochem., 1999, 73, 2460-2468) and scrapie infections (Park etal., NeuroReport, 2000, 11, 1677-1682).

[0008] BCL2-associated X protein knockout mice have been generated andproved viable, but displayed lineage-specific aberrations in cell death(Knudson et al., Science, 1995, 270, 96-99). Additionally, prolongationof ovarian life span into advanced chronological age has been observedin BCL2-associated X protein knockout mice (Perez et al., Nat. Genet.,1999, 21, 200-203).

[0009] Currently there exists a need to identify methods of modulatingapoptosis for the therapeutic treatment of human diseases.

[0010] Antisense oligonucleotides, 20 nucleotides in length, targetingbases 83-102 and 103-122 of human BCL2-associated X protein have beenused to inhibit BCL2-associated X protein in human myeloid cells(Manfredini et al., Antisense Nucleic Acid Drug Dev., 1998, 8, 341-350)and neutrophils (Dibbert et al., Proc. Natl. Acad. Sci. U.S. A., 1999,96, 13330-13335).

[0011] Currently, there remains a need for additional agents capable ofeffectively and selectively inhibiting the expression of BCL2-associatedX protein.

[0012] Modified antisense phosphorothioate oligonucleotides andunmodified oligodeoxynucleotides targeting the start codon of ratBCL2-associated X protein have been used to inhibit BCL2-associated Xprotein in rat neurons (Gillardon et al., J. Neurosci. Res., 1996, 43,726-734; Isenmann et al., Cell Death Differ., 1999, 6, 673-682; Tamataniet al., J. Neurochem., 1998, 71, 1588-1596; Tamatani et al., Cell DeathDiffer., 1998, 5, 911-919) and R6 fibroblasts (Otter et al., J. Biol.Chem., 1998, 273, 6110-6120).

[0013] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of BCL2-associated X proteingene expression and cellular processes.

[0014] The present invention provides compositions and methods formodulating BCL2-associated X protein expression, including modulation ofsplice variants of BCL2-associated X protein including BAX-alpha,BAX-beta, BAX-gamma, BAX-delta, BAX-omega, and BAX-epsilon.

SUMMARY OF THE INVENTION

[0015] The present invention is directed to compounds, particularlyantisense oligonucleotides, which are targeted to a nucleic acidencoding BCL2-associated X protein, and which modulate the expression ofBCL2-associated X protein. Pharmaceutical and other compositionscomprising the compounds of the invention are also provided. Furtherprovided are methods of modulating the expression of BCL2-associated Xprotein in cells or tissues comprising contacting said cells or tissueswith one or more of the antisense compounds or compositions of theinvention. Further provided are methods of treating an animal,particularly a human, suspected of having or being prone to a disease orcondition associated with expression of BCL2-associated X protein byadministering a therapeutically or prophylactically effective amount ofone or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding BCL2-associated X protein, ultimatelymodulating the amount of BCL2-associated X protein produced. This isaccomplished by providing antisense compounds which specificallyhybridize with one or more nucleic acids encoding BCL2-associated Xprotein. As used herein, the terms “target nucleic acid” and “nucleicacid encoding BCL2-associated X protein” encompass DNA encodingBCL2-associated X protein, RNA (including pre-mRNA and mRNA) transcribedfrom such DNA, and also cDNA derived from such RNA. The specifichybridization of an oligomeric compound with its target nucleic acidinterferes with the normal function of the nucleic acid. This modulationof function of a target nucleic acid by compounds which specificallyhybridize to it is generally referred to as “antisense”. The functionsof DNA to be interfered with include replication and transcription. Thefunctions of RNA to be interfered with include all vital functions suchas, for example, translocation of the RNA to the site of proteintranslation, translation of protein from the RNA, splicing of the RNA toyield one or more mRNA species, and catalytic activity which may beengaged in or facilitated by the RNA. The overall effect of suchinterference with target nucleic acid function is modulation of theexpression of BCL2-associated X protein. In the context of the presentinvention, “modulation” means either an increase (stimulation) or adecrease (inhibition) in the expression of a gene. In the context of thepresent invention, inhibition is the preferred form of modulation ofgene expression and mRNA is a preferred target.

[0017] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding BCL2-associated X protein. The targeting process alsoincludes determination of a site or sites within this gene for theantisense interaction to occur such that the desired effect, e.g.,detection or modulation of expression of the protein, will result.Within the context of the present invention, a preferred intragenic siteis the region encompassing the translation initiation or terminationcodon of the open reading frame (ORF) of the gene. Since, as is known inthe art, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon”. A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (in prokaryotes). It is also known in the art thateukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA molecule transcribedfrom a gene encoding BCL2-associated X protein, regardless of thesequence(s) of such codons.

[0018] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0019] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0020] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. It has also beenfound that introns can also be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0021] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0022] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

[0023] Antisense and other compounds of the invention which hybridize tothe target and inhibit expression of the target are identified throughexperimentation, and the sequences of these compounds are hereinbelowidentified as preferred embodiments of the invention. The target sitesto which these preferred sequences are complementary are hereinbelowreferred to as “active sites” and are therefore preferred sites fortargeting. Therefore another embodiment of the invention encompassescompounds which hybridize to these active sites.

[0024] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0025] For use in kits and diagnostics, the antisense compounds of thepresent invention, either alone or in combination with other antisensecompounds or therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

[0026] Expression patterns within cells or tissues treated with one ormore antisense compounds are compared to control cells or tissues nottreated with antisense compounds and the patterns produced are analyzedfor differential levels of gene expression as they pertain, for example,to disease association, signaling pathway, cellular localization,expression level, size, structure or function of the genes examined.These analyses can be performed on stimulated or unstimulated cells andin the presence or absence of other compounds which affect expressionpatterns.

[0027] Examples of methods of gene expression analysis known in the artinclude DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000,480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

[0028] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotide drugs, including ribozymes, have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

[0029] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0030] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about50 nucleobases (i.e. from about 8 to about 50 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides, even more preferably those comprising from about 12 toabout 30 nucleobases. Antisense compounds include ribozymes, externalguide sequence (EGS) oligonucleotides (oligozymes), and other shortcatalytic RNAs or catalytic oligonucleotides which hybridize to thetarget nucleic acid and modulate its expression.

[0031] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

[0032] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0033] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0034] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697 and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0035] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0036] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0037] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0038] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃) —N(CH₃) —CH₂— and —O—N(CH₃) —CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0039] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′′O—CH₂CH₂OCH₃,also known as 2′-O—(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

[0040] A further prefered modification includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring thereby forming a bicyclic sugar moiety. Thelinkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0041] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0042] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

[0043] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

[0044] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. The compounds of the inventioncan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

[0045] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0046] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0047] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0048] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0049] The antisense compounds of the invention are synthesized in vitroand do not include antisense compositions of biological origin, orgenetic vector constructs designed to direct the in vivo synthesis ofantisense molecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0050] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0051] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0052] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0053] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0054] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0055] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of BCL2-associated X protein is treated by administeringantisense compounds in accordance with this invention. The compounds ofthe invention can be utilized in pharmaceutical compositions by addingan effective amount of an antisense compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the antisensecompounds and methods of the invention may also be usefulprophylactically, e.g., to prevent or delay infection, inflammation ortumor formation, for example.

[0056] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding BCL2-associated X protein, enabling sandwich and other assaysto easily be constructed to exploit this fact. Hybridization of theantisense oligonucleotides of the invention with a nucleic acid encodingBCL2-associated X protein can be detected by means known in the art.Such means may include conjugation of an enzyme to the oligonucleotide,radiolabelling of the oligonucleotide or any other suitable detectionmeans. Kits using such detection means for detecting the level ofBCL2-associated X protein in a sample may also be prepared.

[0057] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O—methoxyethylmodification are believed to be particularly useful for oraladministration.

[0058] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Preferred topical formulationsinclude those in which the oligonucleotides of the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Preferred lipids and liposomes include neutral (e.g.dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Oligonucleotides of the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

[0059] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Prefered bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate,. Preferedfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also prefered are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly prefered combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. applications 08/886,829 (filed Jul. 1, 1997), Ser. Nos. 09/108,673(filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624(filed May 21, 1998) and 09/315,298 (filed May 20, 1999) each of whichis incorporated herein by reference in their entirety.

[0060] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0061] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0062] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0063] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0064] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0065] Emulsions

[0066] The compositions of the present invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

[0067] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0068] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0069] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0070] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0071] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0072] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0073] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of reasons of ease of formulation,efficacy from an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0074] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0075] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0076] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C₈-C₁₂) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0077] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0078] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

[0079] Liposomes

[0080] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0081] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0082] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0083] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0084] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranes.As the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0085] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration, liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0086] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0087] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0088] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0089] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0090] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0091] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasome™ (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

[0092] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEES Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765).

[0093] Various liposomes comprising one or more glycolipids are known inthe art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0094] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

[0095] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0096] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0097] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

[0098] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0099] If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0100] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0101] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0102] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0103] Penetration Enhancers

[0104] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0105] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0106] Surfactants: In connection with the present invention,surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. In addition to bile salts and fattyacids, these penetration enhancers include, for example, sodium laurylsulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetylether) (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p.92); and perfluorochemical emulsions, such as FC-43.Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0107] Fatty acids: Various fatty acids and their derivatives which actas penetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁ ₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0108] Bile salts: The physiological role of bile includes thefacilitation of dispersion and absorption of lipids and fat-solublevitamins (Brunton, Chapter 38 in: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, NewYork, 1996, pp. 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus the term “bilesalts” includes any of the naturally occurring components of bile aswell as any of their synthetic derivatives. The bile salts of theinvention include, for example, cholic acid (or its pharmaceuticallyacceptable sodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0109] Chelating Agents: Chelating agents, as used in connection withthe present invention, can be defined as compounds that remove metallicions from solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0110] Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

[0111] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

[0112] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0113] Carriers

[0114] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0115] Excipients

[0116] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

[0117] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0118] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0119] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0120] Other Components

[0121] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0122] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0123] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

[0124] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0125] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀S found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0126] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

[0127] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxyand 2′-Alkoxy Amidites

[0128] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham MA or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, the standard cycle for unmodifiedoligonucleotides was utilized, except the wait step after pulse deliveryof tetrazole and base was increased to 360 seconds.

[0129] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham MA).

[0130] 2′-Fluoro Amidites

[0131] 2′-Fluorodeoxyadenosine Amidites

[0132] 2′-fluoro oligonucleotides were synthesized as describedpreviously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] andU.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, theprotected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine wassynthesized utilizing commercially available9-beta-D-arabinofuranosyladenine as starting material and by modifyingliterature procedures whereby the 2′-alpha-fluoro atom is introduced bya S_(N)2-displacement of a 2′-beta-trityl group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies and standard methods were used to obtain the5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0133] 2′-Fluorodeoxyguanosine

[0134] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0135] 2′-Fluorouridine

[0136] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

[0137] 2′-Fluorodeoxycytidine

[0138] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0139] 2′-O—(2-Methoxyethyl) Modified Amidites2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

[0140]2,2′-Anhydro[1-(Beta-D-Arabinofuranosyl)-5-Methyluridine]

[0141] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60° C. at 1mm Hg for 24 h) to give a solid that was crushed to a light tan powder(57 g, 85% crude yield). The NMR spectrum was consistent with thestructure, contaminated with phenol as its sodium salt (ca. 5%). Thematerial was used as is for further reactions (or it can be purifiedfurther by column chromatography using a gradient of methanol in ethylacetate (10-25%) to give a white solid, mp 222-4° C.).

[0142] 2′-O-Methoxyethyl-5-Methyluridine

[0143] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160° C. After heating for 48 hours at 155-160°C., the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product. Additional material was obtained byreworking impure fractions.

[0144] 2′-O-Methoxyethyl-5′-O-Dimethoxytrityl-5-Methyluridine

[0145] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

[0146]3′-O-Acetyl-2′-O-Methoxyethyl-5′-O-Dimethoxytrityl-5-Methyluridine

[0147] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by TLC by first quenching the TLC sample with the additionof MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

[0148]3′-O-Acetyl-2′-O-Methoxyethyl-5′-O-Dimethoxytrityl-5-Methyl-4-Triazoleuridine

[0149] A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

[0150] 2′-O-Methoxyethyl-5′-O-Dimethoxytrityl-5-Methylcytidine

[0151] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄₀H (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

[0152]N4-Benzoyl-2′-O-methoxyethyl-5′-O-Dimethoxytrityl-5-Methylcytidine

[0153] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, TLC showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

[0154]N4-Benzoyl-2′-O-Methoxyethyl-5′-O-Dimethoxytrityl-5-Methylcytidine-3′-Amidite

[0155]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L) Tetrazole diisopropylamine (7.1g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) wereadded with stirring, under a nitrogen atmosphere. The resulting mixturewas stirred for 20 hours at room temperature (TLC showed the reaction tobe 95% complete). The reaction mixture was extracted with saturatedNaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes wereback-extracted with CH₂Cl₂ (300 mL), and the extracts were combined,dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

[0156] 2′-O-(Aminooxyethyl) Nucleoside Amidites and2′-O-(Dimethylaminooxyethyl) Nucleoside Amidites

[0157] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

[0158] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

[0159] 5′-O-tert-Butyldiphenylsilyl-O²-2′-Anhydro-5-Methyluridine

[0160] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.The solution was concentrated under reduced pressure to a thick oil.This was partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer was dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution was cooled to −10° C. The resultingcrystalline product was collected by filtration, washed with ethyl ether(3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of whitesolid. TLC and NMR were consistent with pure product.

[0161]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-Hydroxyethyl)-5-Methyluridine

[0162] In a 2 L stainless steel, unstirred pressure reactor was addedborane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood andwith manual stirring, ethylene glycol (350 mL, excess) was addedcautiously at first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

[0163]2′-O-([2-Phthalimidoxy)Ethyl]-5′-t-Butyldiphenylsilyl-5-Methyluridine

[0164]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

[0165]5′-O-tert-Butyldiphenylsilyl-2′-O-[(2-Formadoximinooxy)Ethyl]-5-Methyluridine

[0166]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).

[0167]5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-Dimethylaminooxyethyl]-5-Methyluridine

[0168]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO3 solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO3 (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness . The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

[0169] 2′-O-(Dimethylaminooxyethyl)-5-Methyluridine

[0170] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH).This mixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2—O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

[0171] 5′-O-DMT-2′-O-(Dimethylaminooxyethyl)-5-Methyluridine

[0172] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

[0173]5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-Methyluridine-3′-[(2-Cyanoethyl)-N,N-Diisopropylphosphoramidite]

[0174] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

[0175] 2′-(Aminooxyethoxy) Nucleoside Amidites

[0176] 2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

[0177]N2-Isobutyryl-6—O-Diphenylcarbamoyl-2′-O-(2-Ethylacetyl)-5′-O-(4,4′-Dimethoxytrityl)Guanosine-3′-[(2-Cyanoethyl)-N,N-Diisopropylphosphoramidite]

[0178] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 Al 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6—O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′—O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0179] 2′-Dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

[0180] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′—O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

[0181] 2′-O-[2(2-N,N-Dimethylaminoethoxy)Ethyl]-5-Methyl Uridine

[0182] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) isslowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²—,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

[0183] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-Dimethylaminoethoxy)Ethyl)]-5-Methyl Uridine

[0184] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

[0185]5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-Dimethylaminoethoxy)-Ethyl)]-5-MethylUridine-3′-O-(Cyanoethyl-N,N-Diisopropyl)Phosphoramidite

[0186] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2

[0187] Oligonucleotide Synthesis

[0188] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 380B) using standard phosphoramidite chemistrywith oxidation by iodine.

[0189] Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

[0190] Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

[0191] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0192] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0193] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0194] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0195] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0196] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0197] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3

[0198] Oligonucleoside Synthesis

[0199] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethyl-hydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligo-nucleosides, also identified as amide-4 linked oligonucleo-sides,as well as mixed backbone compounds having, for instance, alternatingMMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0200] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0201] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0202] PNA Synthesis

[0203] Peptide nucleic acids (PNAS) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

[0204] Synthesis of Chimeric Oligonucleotides

[0205] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0206] [2′-O-Me]—[2′-Deoxy]—[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

[0207] Chimeric oligonucleotides having 2′′O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligo-nucleotide segments are synthesizedusing an Applied Biosystems automated DNA synthesizer Model 380B, asabove. oligonucleotides are synthesized using the automated synthesizerand 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphor-amidite for the DNAportion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′and 3′ wings. The standard synthesis cycle is modified by increasing thewait step after the delivery of tetrazole and base to 600 s repeatedfour times for RNA and twice for 2′-O-methyl. The fully protectedoligonucleotide is cleaved from the support and the phosphate group isdeprotected in 3:1 ammonia/ethanol at room temperature overnight thenlyophilized to dryness. Treatment in methanolic ammonia for 24 hrs atroom temperature is then done to deprotect all bases and sample wasagain lyophilized to dryness. The pellet is resuspended in 1M TBAF inTHF for 24 hrs at room temperature to deprotect the 2′ positions. Thereaction is then quenched with 1M TEAA and the sample is then reduced to½ volume by rotovac before being desalted on a G25 size exclusioncolumn. The oligo recovered is then analyzed spectrophotometrically foryield and for purity by capillary electrophoresis and by massspectrometry.

[0208] [2′-O-(2-Methoxyethyl)]—[2′-Deoxy]—[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[0209] [2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxy-ethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-0-(methoxyethyl) amidites for the 2′-O-methylamidites.

[0210] [2′-O-(2-Methoxyethyl)Phosphodiesterl]—[2′-DeoxyPhosphorothioate]—[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0211] [2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxyphos-phorothioate]—[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0212] Other chimeric oligonucleotides, chimeric oligonucleo-sides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0213] Oligonucleotide Isolation

[0214] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹p nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

[0215] Oligonucleotide Synthesis -96 Well Plate Format

[0216] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a standard 96 well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites. oligonucleotideswere cleaved from support and deprotected with concentrated NH₄OH atelevated temperature (55-60° C.) for 12-16 hours and the releasedproduct then dried in vacuo. The dried product was then re-suspended insterile water to afford a master plate from which all analytical andtest plate samples are then diluted utilizing robotic pipettors.

Example 8

[0217] Oligonucleotide Analysis -96 Well Plate Format

[0218] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

[0219] Cell Culture and Oligonucleotide Treatment

[0220] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following 5 cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,Ribonuclease protection assays, or RT-PCR.

[0221] T-24 Cells:

[0222] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0223] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0224] A549 Cells:

[0225] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

[0226] NHDF Cells:

[0227] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0228] HEK Cells:

[0229] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville Md.). HEKs were routinely maintainedin Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

[0230] b.END cells:

[0231] The mouse brain endothelial cell line b.END was obtained from Dr.Werner Risau at the Max Plank Instititute (Bad Nauheim, Germany). b.ENDcells were routinely cultured in DMEM, high glucose (Gibco/LifeTechnologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 3000 cells/well for use in RT-PCR analysis.

[0232] For Northern blotting or other analyses, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0233] Treatment With Antisense Compounds:

[0234] When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired concentration of oligonucleotide. After 4-7hours of treatment, the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

[0235] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10

[0236] Analysis of Oligonucleotide Inhibition of BCL2-Associated XProtein Expression

[0237] Antisense modulation of BCL2-associated X protein expression canbe assayed in a variety of ways known in the art. For example,BCL2-associated X protein mRNA levels can be quantitated by, e.g.,Northern blot analysis, competitive polymerase chain reaction (PCR), orreal-time PCR (RT-PCR). Real-time quantitative PCR is presentlypreferred. RNA analysis can be performed on total cellular RNA orpoly(A)+mRNA. Methods of RNA isolation are taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northernblot analysis is routine in the art and is taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative(PCR) can be conveniently accomplished using the commercially availableABI PRISM™ 7700 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

[0238] Protein levels of BCL2-associated X protein can be quantitated ina variety of ways well known in the art, such as immunoprecipitation,Western blot analysis (immunoblotting), ELISA or fluorescence-activatedcell sorting (FACS). Antibodies directed to BCL2-associated X proteincan be identified and obtained from a variety of sources, such as theMSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), orcan be prepared via conventional antibody generation methods. Methodsfor preparation of polyclonal antisera are taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2,pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation ofmonoclonal antibodies is taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5,John Wiley & Sons, Inc., 1997.

[0239] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998. Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11

[0240] Poly(A)+ mRNA Isolation

[0241] Poly(A)+ mRNA was isolated according to Miura et al., Clin.Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolationare taught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C. was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

[0242] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12

[0243] Total RNA Isolation

[0244] Total RNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 100 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 100 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPEwas then added to each well of the RNEASY 96™ plate and the vacuumapplied for a period of 15 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 10 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 60 μL water into each well, incubating1 minute, and then applying the vacuum for 30 seconds. The elution stepwas repeated with an additional 60 μL water.

[0245] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

[0246] Real-Time Quantitative PCR Analysis of BCL2-Associated X ProteinmRNA Levels

[0247] Quantitation of BCL2-associated X protein mRNA levels wasdetermined by real-time quantitative PCR using the ABI PRISM™ 7700Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. This is a closed-tube,non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR, in which amplificationproducts are quantitated after the PCR is completed, products inreal-time quantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., JOE, FAM, orVIC, obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end ofthe probe and a quencher dye (e.g., TAMRA, obtained from either OperonTechnologies Inc., Alameda, Calif. or PE-Applied Biosystems, FosterCity, Calif.) is attached to the 3′ end of the probe. When the probe anddyes are intact, reporter dye emission is quenched by the proximity ofthe 3′ quencher dye. During amplification, annealing of the probe to thetarget sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABIPRISMT™ 7700 Sequence Detection System. In each assay, a series ofparallel reactions containing serial dilutions of mRNA from untreatedcontrol samples generates a standard curve that is used to quantitatethe percent inhibition after antisense oligonucleotide treatment of testsamples.

[0248] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0249] PCR reagents were obtained from PE-Applied Biosystems, FosterCity, Calif. RT-PCR reactions were carried out by adding 25 μL PCRcocktail (1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTPand dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer,and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLtotal RNA solution. The RT reaction was carried out by incubation for 30minutes at 48° C. Following a 10 minute incubation at 95° C. to activatethe AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carriedout: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5minutes (annealing/extension).

[0250] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent from Molecular Probes. Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,Analytical Biochemistry, 1998, 265, 368-374.

[0251] In this assay, 175 μL of RiboGreen working reagent (RiboGreen™reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 25uL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at480 nm and emission at 520 nm.

[0252] Probes and primers to human BCL2-associated X protein weredesigned to hybridize to a human BCL2-associated X protein sequence,using published sequence information (GenBank accession numberNM_(—)004324, incorporated herein as SEQ ID NO:3). For humanBCL2-associated X protein the PCR primers were:

[0253] forward primer: CCGCCGTGGACACAGACT (SEQ ID NO: 4)

[0254] reverse primer: CCGGCCCCAGTTGAAGTT (SEQ ID NO: 5) and the PCR

[0255] probe was: FAM-CCCGAGAGGTCTTTTTCCGAGTGGC-TAMRA

[0256] (SEQ ID NO: 6) where FAM (PE-Applied Biosystems, Foster City,Calif.) is the fluorescent reporter dye) and TAMRA (PE-AppliedBiosystems, Foster City, Calif.) is the quencher dye. For human GAPDHthe PCR primers were:

[0257] forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7)

[0258] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the

[0259] PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO:9) where JOE (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

[0260] Probes and primers to mouse BCL2-associated X protein weredesigned to hybridize to a mouse BCL2-associated X protein sequence,using published sequence information (GenBank accession numberNM_(—)007527, incorporated herein as SEQ ID NO:10). For mouseBCL2-associated X protein the PCR primers were:

[0261] forward primer: AGACACCTGAGCTGACCTTGGA (SEQ ID NO:11)

[0262] reverse primer: GAGACACTCGCTCAGCTTCTTG (SEQ ID NO: 12) and

[0263] the PCR probe was: FAM-AGCCGCCCCAGGATGCGTC-TAMRA

[0264] (SEQ ID NO: 13) where FAM (PE-Applied Biosystems, Foster City,Calif.) is the fluorescent reporter dye) and TAMRA (PE-AppliedBiosystems, Foster City, Calif.) is the quencher dye. For mouse GAPDHthe PCR primers were:

[0265] forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 14)

[0266] reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 15) and the

[0267] PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQID NO: 16) where JOE (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

Example 14

[0268] Northern Blot Analysis of BCL2-Associated X Protein mRNA Levels

[0269] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0270] To detect human BCL2-associated X protein, a humanBCL2-associated X protein specific probe was prepared by PCR using theforward primer CCGCCGTGGACACAGACT (SEQ ID NO: 4) and the reverse primerCCGGCCCCAGTTGAAGTT (SEQ ID NO: 5). To normalize for variations inloading and transfer efficiency membranes were stripped and probed forhuman glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

[0271] To detect mouse BCL2-associated X protein, a mouseBCL2-associated X protein specific probe was prepared by PCR using theforward primer AGACACCTGAGCTGACCTTGGA (SEQ ID NO:11) and the reverseprimer GAGACACTCGCTCAGCTTCTTG (SEQ ID NO: 12). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH)RNA (Clontech, Palo Alto, Calif.).

[0272] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15

[0273] Antisense Inhibition of Human BCL2-Associated X ProteinExpression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap

[0274] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humanBCL2-associated X protein RNA, using published sequences (GenBankaccession number NM_(—)004324 representing the splice variant BAX-betaand incorporated herein as SEQ ID NO: 3, GenBank accession numberAF007826 representing the splice variant BAX-epsilon and incorporatedherein as SEQ ID NO: 17, GenBank accession number L22473 representingthe splice variant BAX-alpha and incorporated herein as SEQ ID NO: 18,GenBank accession number AI382305, the complement of which represents agenomic portion of the splice variant BAX-delta and incorporated hereinas SEQ ID NO: 19, GenBank accession number AF008195 representing agenomic portion of the splice variant BAX-epsilon and incorporatedherein as SEQ ID NO: 20, GenBank accession number AF008196 representingthe splice variant BAX-omega and incorporated herein as SEQ ID NO: 21,and GenBank accession number L22475 representing the splice variantBAX-gamma and incorporated herein as SEQ ID NO: 22). Theoligonucleotides are shown in Table 1. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 1 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humanBCL2-associated X protein mRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from twoexperiments. If present, “N.D.” indicates “no data”. TABLE 1 Inhibitionof human BCL2-associated X protein mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET SEQ ID TARGET SEQ ID ISIS # REGION NO SITE SEQUENCE % INHIB NO134282 Coding 3 561 tccagaaggcccagggtccc 51 23 134283 Coding 3 615tatagaccacatctgatgat 56 24 134284 Coding 3 627 aggaaaacgcattatagacc 5925 134285 Coding 17 6 tgggctgctccccggacccg 80 26 134286 Coding 17 32ctcagagctggtgggccccc 32 27 134287 Coding 17 38 gatctgctcagagctggtgg 5828 134288 Coding 17 50 ccctgtcttcatgatctgct 83 29 134289 Coding 17 51cccctgtcttcatgatctgc 77 30 134290 Coding 17 52 gcccctgtcttcatgatctg 5731 134291 Coding 17 54 gggcccctgtcttcatgatc 17 32 134292 Coding 17 58aaaagggcccctgtcttcat 40 33 134293 Coding 17 70 aaaccctgaagcaaaagggc 5434 134294 Coding 17 71 gaaaccctgaagcaaaaggg 42 35 134295 Coding 17 77ctggatgaaaccctgaagca 15 36 134296 Coding 17 78 cctggatgaaaccctgaagc 4437 134297 Coding 17 79 tcctggatgaaaccctgaag 29 38 134298 Coding 17 82cgatcctggatgaaaccctg 58 39 134299 Coding 17 85 gctcgatcctggatgaaacc 6740 134300 Coding 17 87 ctgctcgatcctggatgaaa 62 41 134301 Coding 17 90gccctgctcgatcctggatg 71 42 134302 Coding 17 146 ggacgcatcctgaggcaccg 7143 134303 Coding 17 147 tggacgcatcctgaggcacc 65 44 134304 Coding 17 155cttcttggtggacgcatcct 69 45 134305 Coding 17 163 tcgctcagcttcttggtgga 3246 134306 Coding 17 167 acactcgctcagcttcttgg 69 47 134307 Coding 17 168gacactcgctcagcttcttg 78 48 134308 Coding 17 170 gagacactcgctcagcttct 5749 134309 Coding 17 209 cagctccatgttactgtcca 71 50 134310 Coding 17 211tgcagctccatgttactgtc 59 51 134311 Coding 17 214 ctctgcagctccatgttact 4852 134312 Coding 17 220 atcatcctctgcagctccat 72 53 134313 Coding 17 224ggcaatcatcctctgcagct 61 54 134314 Coding 17 226 gcggcaatcatcctctgcag 2355 134315 Coding 17 227 ggcggcaatcatcctctgca 30 56 134316 Coding 17 237ctgtgtccacggcggcaatc 76 57 134317 Coding 17 239 gtctgtgtccacggcggcaa 8158 134318 Coding 17 263 tcggaaaaagacctctcggg 73 59 134319 Coding 17 271gctgccactcggaaaaagac 69 60 134320 Coding 17 275 gtcagctgccactcggaaaa 7761 134321 Coding 17 286 tcagaaaacatgtcagctgc 49 62 134322 Coding 17 292ttgccgtcagaaaacatgtc 64 63 134323 Coding 17 305 gccccagttgaagttgccgt 8464 134324 Coding 17 307 cggccccagttgaagttgcc 56 65 134325 Coding 17 331gcaaagtagaaaagggcgac 43 66 134326 Stop 17 485 tggttctgatcagttccggc 51 67Codon 134327 Stop 17 492 cccatgatggttctgatcag 52 68 Codon 134328 3′UTR17 499 tgtccagcccatgatggttc 73 69 134329 3′UTR 17 517ctcccggaggaagtccaatg 71 70 134330 3′UTR 17 521 gccgctcccggaggaagtcc 7271 134331 3′UTR 17 541 gtcttggatccagcccaaca 57 72 134332 3′UTR 17 549ccaccctggtcttggatcca 49 73 134333 3′UTR 17 556 gtcccaaccaccctggtctt 4474 134334 3′UTR 17 564 aggaggccgtcccaaccacc 55 75 134335 3′UTR 17 571gtaggagaggaggccgtccc 67 76 134336 3′UTR 17 605 agatggtcacggtctgccac 7977 134337 3′UTR 17 607 aaagatggtcacggtctgcc 74 78 134338 3′UTR 17 613cgccacaaagatggtcacgg 82 79 134339 3′UTR 17 645 ttccagatggtgagcgaggc 6480 134340 3′UTR 17 647 tcttccagatggtgagcgag 55 81 134341 3′UTR 17 649cttcttccagatggtgagcg 46 82 134342 3′UTR 17 652 catcttcttccagatggtga 5283 134343 3′UTR 17 657 cagcccatcttcttccagat 49 84 134344 Coding 18 359gcacagggccttgagcacca 68 85 134345 3′UTR 19 133 aatgcccatgtcccccaatc 6686 134346 3′UTR 19 182 ctcaagaccacttttcccca 8 87 134347 3′UTR 19 213tttttgggtcccgaaggagg 24 88 134348 Exon 4 20 91 cccaccttgagcaccagttt 7189 134349 Exon 4 20 96 agctgcccaccttgagcacc 68 90 134350 Intron 4 20 261tccagtaaatgcttgttgaa 74 91 134351 Intron 4 20 357 tcacccctgcacgtgaactc67 92 134352 Intron 4 20 483 tgaaccaagatcatgccatt 7 93 134353 Intron 420 497 ggcagaggttgcagtgaacc 58 94 134354 Intron 21 197caaatccgtcttccaaataa 1 95 134355 Intron 21 405 aggttgcgccattgcactcc 5396 134356 Intron 21 490 tggcacatgcctgtaatccc 52 97 134357 Intron 21 511atacaaaattaccgggcgtg 0 98 134358 Intron 21 796 cttggtgcacagggcctgtg 1799 134359 Coding 22 22 gatgaaaccccgcctctggg 35 100

[0275] As shown in Table 1, SEQ ID NOs 23, 24, 25, 26, 28, 29, 30, 31,33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 51, 52, 53,54, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 89, 90, 91, 92, 94,96 and 97 demonstrated at least 40% inhibition of human BCL2-associatedX protein expression in this assay and are therefore preferred. Thetarget sites to which these preferred sequences are complementary areherein referred to as “active sites” and are therefore preferred sitesfor targeting by compounds of the present invention.

Example 16

[0276] Targeting of Individual Oligonucleotides to Specific Variants ofBCL2-Associated X Protein

[0277] In one embodiment of the present invention are oligoncleotidesthat target specific isoforms of BCL2associated X protein. A summary ofthe target sites of the variants ALPHA, BETA, GAMMA, DELTA, EPSILON ANDOMEGA for each individual oligonucleotide is given in Table 2. TABLE 2Targeting of individual oligonucleotides to specific variants ofBCL2—associated X protein SEQ ID TARGET ISIS # NO SITE VARIANT TARGETSEQ ID 134282 23 561 BETA 3 134283 24 615 BETA 3 134284 25 627 BETA 3134285 26 6 LPHA 18 134285 26 6 BETA 3 134285 26 6 GAMMA 22 134285 26 6EPSILON 17 134286 27 32 ALPHA 18 134286 27 32 BETA 3 134286 27 32EPSILON 17 134287 28 38 ALPHA 18 134287 28 38 BETA 3 134287 28 38EPSILON 17 134288 29 50 ALPHA 18 134288 29 50 BETA 3 134288 29 50EPSILON 17 134289 30 51 ALPHA 18 134289 30 51 BETA 3 134289 30 51EPSILON 17 134290 31 52 ALPHA 18 134290 31 52 BETA 3 134290 31 52EPSILON 17 134291 32 54 ALPHA 18 134291 32 54 BETA 3 134291 32 54EPSILON 17 134292 33 58 ALPHA 18 134292 33 58 BETA 3 134292 33 58EPSILON 17 134293 34 70 ALPHA 18 134293 34 70 BETA 3 134293 34 70EPSILON 17 134294 35 71 ALPHA 18 134294 35 71 BETA 3 134294 35 71EPSILON 17 134295 36 77 ALPHA 18 134295 36 77 BETA 3 134295 36 77EPSILON 17 134296 37 78 ALPHA 18 134296 37 78 BETA 3 134296 37 78EPSILON 17 134297 38 79 ALPHA 18 134297 38 79 BETA 3 134297 38 79EPSILON 17 134298 39 82 ALPHA 18 134298 39 82 BETA 3 134298 39 82EPSILON 17 134299 40 85 ALPHA 18 134299 40 85 BETA 3 134299 40 33 GAMMA22 134299 40 85 EPSILON 17 134300 41 87 ALPHA 18 134300 41 87 BETA 3134300 41 35 GAMMA 22 134300 41 87 EPSILON 17 134301 42 90 ALPHA 18134301 42 90 BETA 3 134301 42 38 GAMMA 22 134301 42 90 EPSILON 17 13430243 146 ALPHA 18 134302 43 146 BETA 3 134302 43 94 GAMMA 22 134302 43 146EPSILON 17 134303 44 147 ALPHA 18 134303 44 147 BETA 3 134303 44 95GAMMA 22 134303 44 147 EPSILON 17 134304 45 155 ALPHA 18 134304 45 155BETA 3 134304 45 103 GAMMA 22 134304 45 155 EPSILON 17 134305 46 163ALPHA 18 134305 46 163 BETA 3 134305 46 163 EPSILON 17 134306 47 167ALPHA 18 134306 47 167 BETA 3 134306 47 167 EPSILON 17 134307 48 168ALPHA 18 134307 48 168 BETA 3 134307 48 168 EPSILON 17 134308 49 170ALPHA 18 134308 49 170 BETA 3 134308 49 170 EPSILON 17 134309 50 209ALPHA 18 134309 50 209 BETA 3 134309 50 209 EPSILON 17 134310 51 211ALPHA 18 134310 51 211 BETA 3 134310 51 211 EPSILON 17 134311 52 214ALPHA 18 134311 52 214 BETA 3 134311 52 214 EPSILON 17 134312 53 220ALPHA 18 134312 53 220 BETA 3 134312 53 220 EPSILON 17 134313 54 224ALPHA 18 134313 54 224 BETA 3 134313 54 224 EPSILON 17 134314 55 226ALPHA 18 134314 55 226 BETA 3 134314 55 226 EPSILON 17 134315 56 227ALPHA 18 134315 56 227 BETA 3 134315 56 227 EPSILON 17 134316 57 237ALPHA 18 134316 57 237 BETA 3 134316 57 237 EPSILON 17 134317 58 239ALPHA 18 134317 58 239 BETA 3 134317 58 239 EPSILON 17 134318 59 263ALPHA 18 134318 59 263 BETA 3 134318 59 263 EPSILON 17 134319 60 271ALPHA 18 134319 60 271 BETA 3 134319 60 271 EPSILON 17 134319 60 7EPSILON 20 134320 61 275 ALPHA 18 134320 61 275 BETA 3 134320 61 275EPSILON 17 134320 61 11 EPSILON 20 134321 62 286 ALPHA 18 134321 62 286BETA 3 134321 62 286 EPSILON 17 134321 62 22 EPSILON 20 134322 63 292ALPHA 18 134322 63 292 BETA 3 134322 63 292 EPSILON 17 134322 63 28EPSILON 20 134323 64 305 ALPHA 18 134323 64 305 BETA 3 134323 64 305EPSILON 17 134323 64 41 EPSILON 20 134324 65 307 ALPHA 18 134324 65 307BETA 3 134324 65 307 EPSILON 17 134324 65 43 EPSILON 20 134325 66 331ALPHA 18 134325 66 331 BETA 3 134325 66 331 EPSILON 17 134325 66 67EPSILON 20 134326 67 387 ALPHA 18 134326 67 387 BETA 3 134326 67 485EPSILON 17 134327 68 394 ALPHA 18 134327 68 394 BETA 3 134327 68 492EPSILON 17 134328 69 401 ALPHA 18 134328 69 401 BETA 3 134328 69 499EPSILON 17 134329 70 419 ALPHA 18 134329 70 419 BETA 3 134329 70 517EPSILON 17 134330 71 423 ALPHA 18 134330 71 423 BETA 3 134330 71 521EPSILON 17 134331 72 443 ALPHA 18 134331 72 443 BETA 3 134331 72 541EPSILON 17 134332 73 451 ALPHA 18 134332 73 451 BETA 3 134332 73 549EPSILON 17 134333 74 458 ALPHA 18 134333 74 556 EPSILON 17 134334 75 466ALPHA 18 134334 75 564 EPSILON 17 134335 76 473 ALPHA 18 134335 76 571EPSILON 17 134336 77 507 ALPHA 18 134336 77 605 EPSILON 17 134337 78 509ALPHA 18 134337 78 607 EPSILON 17 134338 79 515 ALPHA 18 134338 79 613EPSILON 17 134339 80 547 ALPHA 18 134339 80 645 EPSILON 17 134340 81 549ALPHA 18 134340 81 647 EPSILON 17 134341 82 551 ALPHA 18 134341 82 649EPSILON 17 134342 83 554 ALPHA 18 134342 83 652 EPSILON 17 134343 84 559ALPHA 18 134343 84 657 EPSILON 17 134344 85 359 ALPHA 18 134344 85 359BETA 3 134345 86 133 DELTA 19 134346 87 182 DELTA 19 134347 88 213 DELTA19 134348 89 91 EPSILON 20 134349 90 96 EPSILON 20 134350 91 261 EPSILON20 134351 92 357 EPSILON 20 134352 93 483 EPSILON 20 134353 94 497EPSILON 20 134354 95 197 OMEGA 21 134355 96 405 OMEGA 21 134356 97 490OMEGA 21 134357 98 511 OMEGA 21 134358 99 796 OMEGA 21 134359 100 22GAMMA 22

Example 17

[0278] Antisense Inhibition of Mouse BCL2-Associated X ProteinExpression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap.

[0279] In accordance with the present invention, a second series ofoligonucleotides were designed to target different regions of the mouseBCL2-associated X protein RNA, using published sequences (GenBankaccession number NM_(—)007527, incorporated herein as SEQ ID NO: 10,GenBank accession number AB029557, incorporated herein as SEQ ID NO:101, and GenBank accession number AA104861, an EST suggesting a splicevariant that diverges at nucleotide 34 of GenBank accession numberNM_(—)007527, incorporated herein as SEQ ID NO: 102). Theoligonucleotides are shown in Table 3. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 3 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mouseBCL2-associated X protein mRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from twoexperiments. If present, “N.D.” indicates “no data”.

Table 3

[0280] Inhibition of mouse BCL2-associated X protein mRNA levels bychimeric phosphorothioate oligonucleotides having 2′-MOE wings and adeoxy gap TARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITE SEQUENCE %INHIB NO 134290 Coding 10 52 gcccctgtcttcatgatctg 43 31 134298 Coding 1082 cgatcctggatgaaaccctg 56 39 134300 Coding 10 87 ctgctcgatcctggatgaaa54 41 134305 Coding 10 163 tcgctcagcttcttggtgga 70 46 134307 Coding 10168 gacactcgctcagcttcttg 66 48 134312 Coding 10 220 atcatcctctgcagctccat32 53 134324 Coding 10 307 cggccccagttgaagttgcc 49 65 134327 Coding 10394 cccatgatggttctgatcag 64 68 134328 Coding 10 401 tgtccagcccatgatggttc16 69 134332 Coding 10 451 ccaccctggtcttggatcca 16 73 134342 Coding 10554 catcttcttccagatggtga 58 83 134343 Stop 10 559 cagcccatcttcttccagat76 84 Codon 134382 Coding 10 49 cctgtcttcatgatctgttc 48 103 134383Coding 10 109 ggtgtctccccagccatcct 68 104 134384 Coding 10 116cagctcaggtgtctccccag 61 105 134385 Coding 10 123 ccaaggtcagctcaggtgtc 64106 134386 Coding 10 129 gctgctccaaggtcagctca 38 107 134387 Coding 10134 gggcggctgctccaaggtca 34 108 134388 Coding 10 181ccaattcgccggagacactc 49 109 134389 Coding 10 212 ctgcagctccatattgctat 62110 134390 Coding 10 226 tcagcaatcatcctctgcag 51 111 134391 Coding 10231 ccacgtcagcaatcatcctc 70 112 134392 Coding 10 238tccgtgtccacgtcagcaat 63 113 134393 Coding 10 242 ggagtccgtgtccacgtcag 49114 134394 Coding 10 269 tgccacccggaagaagacct 49 115 134395 Coding 10283 gcaaacatgtcagctgccac 51 116 134396 Coding 10 292ttgccatcagcaaacatgtc 45 117 134397 Coding 10 300 agttgaagttgccatcagca 57118 134398 Coding 10 303 cccagttgaagttgccatca 42 119 134399 Coding 10312 ccacgcggccccagttgaag 3 120 134400 Coding 10 340 agtttgctagcaaagtagaa43 121 134401 Coding 10 348 tgagcaccagtttgctagca 71 122 134402 Coding 10357 acagggccttgagcaccagt 31 123 134403 Coding 10 363tagtgcacagggccttgagc 43 124 134404 Coding 10 365 tttagtgcacagggccttga 58125 134405 Coding 10 374 ctcgggcactttagtgcaca 67 126 134406 Coding 10376 agctcgggcactttagtgca 50 127 134407 Coding 10 380gatcagctcgggcactttag 18 128 134408 Coding 10 387 tggttctgatcagctcgggc 49129 134409 Coding 10 393 ccatgatggttctgatcagc 40 130 134410 Coding 10409 aagtccagtgtccagcccat 54 131 134411 Coding 10 412aggaagtccagtgtccagcc 73 132 134412 Coding 10 415 cggaggaagtccagtgtcca 16133 134413 Coding 10 437 gatccagacaagcagccgct 37 134 134414 Coding 10445 tggtcttggatccagacaag 39 135 134415 Coding 10 457tcccagccaccctggtcttg 71 136 134416 Coding 10 505 atggtcactgtctgccatgt 61137 134417 Coding 10 508 aagatggtcactgtctgcca 62 138 134418 Coding 10514 gccacaaagatggtcactgt 67 139 134419 Coding 10 523aggactccagccacaaagat 54 140 134420 Coding 10 533 cgaggcggtgaggactccag 46141 134421 Coding 10 546 tccagatggtgagcgaggcg 48 142 134422 Coding 10550 ttcttccagatggtgagcga 57 143 134423 Stop 10 560 tcagcccatcttcttccaga54 144 Codon 134424 5′UTR 101 233 gcaggaccatctctctgagt 53 145 1344255′UTR 101 264 tggtttgaaacctagcacac 32 146 134426 5′UTR 101 338agatatgtccctgggccttg 48 147 134427 5′UTR 101 447 aaactagcacactgcttgac 16148 134428 5′UTR 101 498 cctaccttggtttcccaaga 32 149 134429 5′UTR 101655 gctgggattaaaggcgtgcg 46 150 134430 5′UTR 101 682aaatccgcctgcctctgcct 31 151 134431 5′UTR 101 896 ggcctccaacatagagaacc 21152 134432 5′UTR 101 1032 atctgggtgtcagaaagcct 28 153 134433 5′UTR 1011303 cgcaggaaagtagatctctg 24 154 134434 5′UTR 101 1562agcaagtgacaggtcaatta 22 155 134435 5′UTR 101 2077 aaacaggctgtacgcggtgg19 156 134436 5′UTR 101 2210 agttgaccagagtgatggtc 38 157 134437 5′UTR101 2530 gtcacgtgatcatcatcgct 47 158 134438 Start 101 2666ggacccgtccatcactgccg 55 159 Codon 134439 Coding 102 107actgcttctgatggacaggg 64 160 134440 Coding 102 116 ggcctggctactgcttctga21 161 134441 Coding 102 124 gcatggaaggcctggctact 41 162 134442 Coding102 137 gtagtgacaagtagcatgga 60 163 134443 Coding 102 143accctagtagtgacaagtag 57 164 134444 Coding 102 178 taggctcataaccctgaggg46 165 134445 Coding 102 187 atggataggtaggctcataa 48 166 134446 Coding102 220 ctggactcctgggtcccagg 58 167 134447 Coding 102 263tcagagctggtgggccctgg 62 168

[0281] As shown in Table 3, SEQ ID NOs 31, 39, 41, 46, 48, 65, 68, 83,84, 103, 104, 105, 106, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 121, 122, 124, 125, 126, 127, 129, 130, 131, 132, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 147, 150, 158, 159, 160, 162,163, 164, 165, 166, 167 and 168 demonstrated at least 40% inhibition ofmouse BCL2-associated X protein expression in this experiment and aretherefore preferred. The target sites to which these preferred sequencesare complementary are herein referred to as “active sites” and aretherefore preferred sites for targeting by compounds of the presentinvention.

Example 18

[0282] Western Blot Analysis of BCL2-Associated X Protein Protein Levels

[0283] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to BCL2-associated Xprotein is used, with a radiolabelled or fluorescently labeled secondaryantibody directed against the primary antibody species. Bands arevisualized using a PHOSPHORIMAGER™ (Molecular Dynamics, SunnyvaleCalif.).

1 168 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence AntisenseOligonucleotide 2 atgcattctg cccccaagga 20 3 657 DNA Homo sapiens CDS(1)...(657) 3 atg gac ggg tcc ggg gag cag ccc aga ggc ggg ggg ccc accagc tct 48 Met Asp Gly Ser Gly Glu Gln Pro Arg Gly Gly Gly Pro Thr SerSer 1 5 10 15 gag cag atc atg aag aca ggg gcc ctt ttg ctt cag ggt ttcatc cag 96 Glu Gln Ile Met Lys Thr Gly Ala Leu Leu Leu Gln Gly Phe IleGln 20 25 30 gat cga gca ggg cga atg ggg ggg gag gca ccc gag ctg gcc ctggac 144 Asp Arg Ala Gly Arg Met Gly Gly Glu Ala Pro Glu Leu Ala Leu Asp35 40 45 ccg gtg cct cag gat gcg tcc acc aag aag ctg agc gag tgt ctc aag192 Pro Val Pro Gln Asp Ala Ser Thr Lys Lys Leu Ser Glu Cys Leu Lys 5055 60 cgc atc ggg gac gaa ctg gac agt aac atg gag ctg cag agg atg att240 Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Glu Leu Gln Arg Met Ile 6570 75 80 gcc gcc gtg gac aca gac tcc ccc cga gag gtc ttt ttc cga gtg gca288 Ala Ala Val Asp Thr Asp Ser Pro Arg Glu Val Phe Phe Arg Val Ala 8590 95 gct gac atg ttt tct gac ggc aac ttc aac tgg ggc cgg gtt gtc gcc336 Ala Asp Met Phe Ser Asp Gly Asn Phe Asn Trp Gly Arg Val Val Ala 100105 110 ctt ttc tac ttt gcc agc aaa ctg gtg ctc aag gcc ctg tgc acc aag384 Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Lys Ala Leu Cys Thr Lys 115120 125 gtg ccg gaa ctg atc aga acc atc atg ggc tgg aca ttg gac ttc ctc432 Val Pro Glu Leu Ile Arg Thr Ile Met Gly Trp Thr Leu Asp Phe Leu 130135 140 cgg gag cgg ctg ttg ggc tgg atc caa gac cag ggt ggt tgg gtg aga480 Arg Glu Arg Leu Leu Gly Trp Ile Gln Asp Gln Gly Gly Trp Val Arg 145150 155 160 ctc ctc aag cct cct cac ccc cac cac cgc gcc ctc acc acc gcccct 528 Leu Leu Lys Pro Pro His Pro His His Arg Ala Leu Thr Thr Ala Pro165 170 175 gcc cca ccg tcc ctg ccc ccc gcc act cct ctg gga ccc tgg gccttc 576 Ala Pro Pro Ser Leu Pro Pro Ala Thr Pro Leu Gly Pro Trp Ala Phe180 185 190 tgg agc agg tca cag tgg tgc cct ctc ccc atc ttc aga tca tcagat 624 Trp Ser Arg Ser Gln Trp Cys Pro Leu Pro Ile Phe Arg Ser Ser Asp195 200 205 gtg gtc tat aat gcg ttt tcc tta cgt gtc tga 657 Val Val TyrAsn Ala Phe Ser Leu Arg Val 210 215 4 18 DNA Artificial Sequence PCRPrimer 4 ccgccgtgga cacagact 18 5 18 DNA Artificial Sequence PCR Primer5 ccggccccag ttgaagtt 18 6 25 DNA Artificial Sequence PCR Probe 6cccgagaggt ctttttccga gtggc 25 7 19 DNA Artificial Sequence PCR Primer 7gaaggtgaag gtcggagtc 19 8 20 DNA Artificial Sequence PCR Primer 8gaagatggtg atgggatttc 20 9 20 DNA Artificial Sequence PCR Probe 9caagcttccc gttctcagcc 20 10 579 DNA Mus musculus CDS (1)...(579) 10 atggac ggg tcc ggg gag cag ctt ggg agc ggc ggg ccc acc agc tct 48 Met AspGly Ser Gly Glu Gln Leu Gly Ser Gly Gly Pro Thr Ser Ser 1 5 10 15 gaacag atc atg aag aca ggg gcc ttt ttg cta cag ggt ttc atc cag 96 Glu GlnIle Met Lys Thr Gly Ala Phe Leu Leu Gln Gly Phe Ile Gln 20 25 30 gat cgagca ggg agg atg gct ggg gag aca cct gag ctg acc ttg gag 144 Asp Arg AlaGly Arg Met Ala Gly Glu Thr Pro Glu Leu Thr Leu Glu 35 40 45 cag ccg ccccag gat gcg tcc acc aag aag ctg agc gag tgt ctc cgg 192 Gln Pro Pro GlnAsp Ala Ser Thr Lys Lys Leu Ser Glu Cys Leu Arg 50 55 60 cga att gga gatgaa ctg gat agc aat atg gag ctg cag agg atg att 240 Arg Ile Gly Asp GluLeu Asp Ser Asn Met Glu Leu Gln Arg Met Ile 65 70 75 80 gct gac gtg gacacg gac tcc ccc cga gag gtc ttc ttc cgg gtg gca 288 Ala Asp Val Asp ThrAsp Ser Pro Arg Glu Val Phe Phe Arg Val Ala 85 90 95 gct gac atg ttt gctgat ggc aac ttc aac tgg ggc cgc gtg gtt gcc 336 Ala Asp Met Phe Ala AspGly Asn Phe Asn Trp Gly Arg Val Val Ala 100 105 110 ctc ttc tac ttt gctagc aaa ctg gtg ctc aag gcc ctg tgc act aaa 384 Leu Phe Tyr Phe Ala SerLys Leu Val Leu Lys Ala Leu Cys Thr Lys 115 120 125 gtg ccc gag ctg atcaga acc atc atg ggc tgg aca ctg gac ttc ctc 432 Val Pro Glu Leu Ile ArgThr Ile Met Gly Trp Thr Leu Asp Phe Leu 130 135 140 cgt gag cgg ctg cttgtc tgg atc caa gac cag ggt ggc tgg gaa ggc 480 Arg Glu Arg Leu Leu ValTrp Ile Gln Asp Gln Gly Gly Trp Glu Gly 145 150 155 160 ctc ctc tcc tacttc ggg acc ccc aca tgg cag aca gtg acc atc ttt 528 Leu Leu Ser Tyr PheGly Thr Pro Thr Trp Gln Thr Val Thr Ile Phe 165 170 175 gtg gct gga gtcctc acc gcc tcg ctc acc atc tgg aag aag atg ggc 576 Val Ala Gly Val LeuThr Ala Ser Leu Thr Ile Trp Lys Lys Met Gly 180 185 190 tga 579 11 22DNA Artificial Sequence PCR Primer 11 agacacctga gctgaccttg ga 22 12 22DNA Artificial Sequence PCR Primer 12 gagacactcg ctcagcttct tg 22 13 19DNA Artificial Sequence PCR Probe 13 agccgcccca ggatgcgtc 19 14 20 DNAArtificial Sequence PCR Primer 14 ggcaaattca acggcacagt 20 15 20 DNAArtificial Sequence PCR Primer 15 gggtctcgct cctggaagat 20 16 27 DNAArtificial Sequence PCR Probe 16 aaggccgaga atgggaagct tgtcatc 27 17 677DNA Homo sapiens CDS (1)...(495) 17 atg gac ggg tcc ggg gag cag ccc agaggc ggg ggg ccc acc agc tct 48 Met Asp Gly Ser Gly Glu Gln Pro Arg GlyGly Gly Pro Thr Ser Ser 1 5 10 15 gag cag atc atg aag aca ggg gcc cttttg ctt cag ggt ttc atc cag 96 Glu Gln Ile Met Lys Thr Gly Ala Leu LeuLeu Gln Gly Phe Ile Gln 20 25 30 gat cga gca ggg cga atg ggg ggg gag gcaccc gag ctg gcc ctg gac 144 Asp Arg Ala Gly Arg Met Gly Gly Glu Ala ProGlu Leu Ala Leu Asp 35 40 45 ccg gtg cct cag gat gcg tcc acc aag aag ctgagc gag tgt ctc aag 192 Pro Val Pro Gln Asp Ala Ser Thr Lys Lys Leu SerGlu Cys Leu Lys 50 55 60 cgc atc ggg gac gaa ctg gac agt aac atg gag ctgcag agg atg att 240 Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Glu Leu GlnArg Met Ile 65 70 75 80 gcc gcc gtg gac aca gac tcc ccc cga gag gtc tttttc cga gtg gca 288 Ala Ala Val Asp Thr Asp Ser Pro Arg Glu Val Phe PheArg Val Ala 85 90 95 gct gac atg ttt tct gac ggc aac ttc aac tgg ggc cgggtt gtc gcc 336 Ala Asp Met Phe Ser Asp Gly Asn Phe Asn Trp Gly Arg ValVal Ala 100 105 110 ctt ttc tac ttt gcc agc aaa ctg gtg ctc aag gct ggcgtg aaa tgg 384 Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Lys Ala Gly ValLys Trp 115 120 125 cgt gat ctg ggc tca ctg caa cct ctg cct cct ggg ttcaag cga ttc 432 Arg Asp Leu Gly Ser Leu Gln Pro Leu Pro Pro Gly Phe LysArg Phe 130 135 140 acc tgc ctc agc atc cca agg agc tgg gat tac agg ccctgt gca cca 480 Thr Cys Leu Ser Ile Pro Arg Ser Trp Asp Tyr Arg Pro CysAla Pro 145 150 155 160 agg tgc cgg aac tga tcagaaccat catgggctggacattggact tcctccggga 535 Arg Cys Arg Asn 165 gcggctgttg ggctggatccaagaccaggg tggttgggac ggcctcctct cctactttgg 595 gacgcccacg tggcagaccgtgaccatctt tgtggcggga gtgctcaccg cctcgctcac 655 catctggaag aagatgggct ga677 18 579 DNA Homo sapiens CDS (1)...(579) 18 atg gac ggg tcc ggg gagcag ccc aga ggc ggg ggg ccc acc agc tct 48 Met Asp Gly Ser Gly Glu GlnPro Arg Gly Gly Gly Pro Thr Ser Ser 1 5 10 15 gag cag atc atg aag acaggg gcc ctt ttg ctt cag ggt ttc atc cag 96 Glu Gln Ile Met Lys Thr GlyAla Leu Leu Leu Gln Gly Phe Ile Gln 20 25 30 gat cga gca ggg cga atg gggggg gag gca ccc gag ctg gcc ctg gac 144 Asp Arg Ala Gly Arg Met Gly GlyGlu Ala Pro Glu Leu Ala Leu Asp 35 40 45 ccg gtg cct cag gat gcg tcc accaag aag ctg agc gag tgt ctc aag 192 Pro Val Pro Gln Asp Ala Ser Thr LysLys Leu Ser Glu Cys Leu Lys 50 55 60 cgc atc ggg gac gaa ctg gac agt aacatg gag ctg cag agg atg att 240 Arg Ile Gly Asp Glu Leu Asp Ser Asn MetGlu Leu Gln Arg Met Ile 65 70 75 80 gcc gcc gtg gac aca gac tcc ccc cgagag gtc ttt ttc cga gtg gca 288 Ala Ala Val Asp Thr Asp Ser Pro Arg GluVal Phe Phe Arg Val Ala 85 90 95 gct gac atg ttt tct gac ggc aac ttc aactgg ggc cgg gtt gtc gcc 336 Ala Asp Met Phe Ser Asp Gly Asn Phe Asn TrpGly Arg Val Val Ala 100 105 110 ctt ttc tac ttt gcc agc aaa ctg gtg ctcaag gcc ctg tgc acc aag 384 Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu LysAla Leu Cys Thr Lys 115 120 125 gtg ccg gaa ctg atc aga acc atc atg ggctgg aca ttg gac ttc ctc 432 Val Pro Glu Leu Ile Arg Thr Ile Met Gly TrpThr Leu Asp Phe Leu 130 135 140 cgg gag cgg ctg ttg ggc tgg atc caa gaccag ggt ggt tgg gac ggc 480 Arg Glu Arg Leu Leu Gly Trp Ile Gln Asp GlnGly Gly Trp Asp Gly 145 150 155 160 ctc ctc tcc tac ttt ggg acg ccc acgtgg cag acc gtg acc atc ttt 528 Leu Leu Ser Tyr Phe Gly Thr Pro Thr TrpGln Thr Val Thr Ile Phe 165 170 175 gtg gcg gga gtg ctc acc gcc tcg ctcacc atc tgg aag aag atg ggc 576 Val Ala Gly Val Leu Thr Ala Ser Leu ThrIle Trp Lys Lys Met Gly 180 185 190 tga 579 19 263 DNA Homo sapiens 19accgtgacca tctttgtggc gggagtgctc cccccctcac tccccatttg gaaaaaaatg 60ggctgaggcc cccagctgcc ttggactgtg tttttccccc ataaattatg gcattttttt 120gggaggggtg gggattgggg gacatgggca tttttcttac ttttgtaatt attggggggt 180gtggggaaaa gtggtcttga gggggtaata aacctccttc gggacccaaa aaaaaaaaaa 240aaaaaaaaaa aaaaaaaaaa aaa 263 20 531 DNA Homo sapiens 20 cgagaggtctttttccgagt ggcagctgac atgttttctg acggcaactt caactggggc 60 cgggttgtcgcccttttcta ctttgccagc aaactggtgc tcaaggtggg cagctgcagg 120 gcagtgagcccagggatgct ccccctcaga tctgtgagga cctggggatc gtggtatcaa 180 ccccctgcagtggcccagtg accacagagg gcatggagag agatggctgt gcactgggtg 240 tctgctccttcttttattca ttcaacaagc atttactgga cctgctatgt gccaggccta 300 tacctggcacctgggacaca gcactgtaca aagcaggcta catccctgct ctcagggagt 360 tcacgtgcaggggtgaagta aagtgggcag agtgatttag cagagtggac aggaaagatt 420 tctatttttttttttttttt ttttgagatg gagttttgct cttgttgccc aggcttgagt 480 gcaatggcatgatcttggtt cactgcaacc tctgcctccc aggttcaagc g 531 21 822 DNA Homosapiens 21 gagaggccta aaaggggagg agtcgggggg gggcgaccga aacatgaagtggaaaggtgg 60 ggtcaggcca aggcgaggca acaaggggtt gggggggcac agtgctggttcttatcgggg 120 gtaaggggag ccacagaggg tcaggggggg ggcagttgga gagtaacaatcttgttgaca 180 attttatgtt ttatatttat ttggaagacg gatttgctta tctccaaggctggcgtgaaa 240 tggcgtgatc tgggctcact gcaacctctg cctcctgggt tcaagcgattcacctgcctc 300 agcatcccaa ggagctggga ttacaggtgc ctgccaccac acccagctaatttttgtatt 360 tatttatttt agagatggag ttttgctctt gttgtgccca ggctggagtgcaatggcgca 420 acctcggctc actgcaacct ccgcctcccg ggttcaagca attctcctgcctcagactcc 480 caagtagctg ggattacagg catgtgccac cacgcccggt aattttgtatttttagtaga 540 gatggcatta ctcccgtaat tggtcaggct ggttttgaat ccggacttcaagtgattccg 600 ctgccttggc cttcccaaag tgctgggatt acaggcatga gccgccgcacctggccatgt 660 ttacaatttt tgaagccgat tcaattgtgg gtggcagaaa ttttgaggggaggcaaagaa 720 ttgacaaagg aggtttgggg ccactatctc aggcagtggg gacaaggttcagtccctaac 780 gcccactcca ctccccacag gccctgtgca ccaaggtgcc gg 822 22 126DNA Homo sapiens CDS (1)...(126) 22 atg gac ggg tcc ggg gag cag ccc agaggc ggg gtt tca tcc agg atc 48 Met Asp Gly Ser Gly Glu Gln Pro Arg GlyGly Val Ser Ser Arg Ile 1 5 10 15 gag cag ggc gaa tgg ggg ggg agg cacccg agc tgg ccc tgg acc cgg 96 Glu Gln Gly Glu Trp Gly Gly Arg His ProSer Trp Pro Trp Thr Arg 20 25 30 tgc ctc agg atg cgt cca cca aga agc tga126 Cys Leu Arg Met Arg Pro Pro Arg Ser 35 40 23 20 DNA ArtificialSequence Antisense Oligonucleotide 23 tccagaaggc ccagggtccc 20 24 20 DNAArtificial Sequence Antisense Oligonucleotide 24 tatagaccac atctgatgat20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 aggaaaacgcattatagacc 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26tgggctgctc cccggacccg 20 27 20 DNA Artificial Sequence AntisenseOligonucleotide 27 ctcagagctg gtgggccccc 20 28 20 DNA ArtificialSequence Antisense Oligonucleotide 28 gatctgctca gagctggtgg 20 29 20 DNAArtificial Sequence Antisense Oligonucleotide 29 ccctgtcttc atgatctgct20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 cccctgtcttcatgatctgc 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31gcccctgtct tcatgatctg 20 32 20 DNA Artificial Sequence AntisenseOligonucleotide 32 gggcccctgt cttcatgatc 20 33 20 DNA ArtificialSequence Antisense Oligonucleotide 33 aaaagggccc ctgtcttcat 20 34 20 DNAArtificial Sequence Antisense Oligonucleotide 34 aaaccctgaa gcaaaagggc20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 gaaaccctgaagcaaaaggg 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36ctggatgaaa ccctgaagca 20 37 20 DNA Artificial Sequence AntisenseOligonucleotide 37 cctggatgaa accctgaagc 20 38 20 DNA ArtificialSequence Antisense Oligonucleotide 38 tcctggatga aaccctgaag 20 39 20 DNAArtificial Sequence Antisense Oligonucleotide 39 cgatcctgga tgaaaccctg20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 gctcgatcctggatgaaacc 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41ctgctcgatc ctggatgaaa 20 42 20 DNA Artificial Sequence AntisenseOligonucleotide 42 gccctgctcg atcctggatg 20 43 20 DNA ArtificialSequence Antisense Oligonucleotide 43 ggacgcatcc tgaggcaccg 20 44 20 DNAArtificial Sequence Antisense Oligonucleotide 44 tggacgcatc ctgaggcacc20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 cttcttggtggacgcatcct 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46tcgctcagct tcttggtgga 20 47 20 DNA Artificial Sequence AntisenseOligonucleotide 47 acactcgctc agcttcttgg 20 48 20 DNA ArtificialSequence Antisense Oligonucleotide 48 gacactcgct cagcttcttg 20 49 20 DNAArtificial Sequence Antisense Oligonucleotide 49 gagacactcg ctcagcttct20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 cagctccatgttactgtcca 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51tgcagctcca tgttactgtc 20 52 20 DNA Artificial Sequence AntisenseOligonucleotide 52 ctctgcagct ccatgttact 20 53 20 DNA ArtificialSequence Antisense Oligonucleotide 53 atcatcctct gcagctccat 20 54 20 DNAArtificial Sequence Antisense Oligonucleotide 54 ggcaatcatc ctctgcagct20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 gcggcaatcatcctctgcag 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56ggcggcaatc atcctctgca 20 57 20 DNA Artificial Sequence AntisenseOligonucleotide 57 ctgtgtccac ggcggcaatc 20 58 20 DNA ArtificialSequence Antisense Oligonucleotide 58 gtctgtgtcc acggcggcaa 20 59 20 DNAArtificial Sequence Antisense Oligonucleotide 59 tcggaaaaag acctctcggg20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 gctgccactcggaaaaagac 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61gtcagctgcc actcggaaaa 20 62 20 DNA Artificial Sequence AntisenseOligonucleotide 62 tcagaaaaca tgtcagctgc 20 63 20 DNA ArtificialSequence Antisense Oligonucleotide 63 ttgccgtcag aaaacatgtc 20 64 20 DNAArtificial Sequence Antisense Oligonucleotide 64 gccccagttg aagttgccgt20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 cggccccagttgaagttgcc 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66gcaaagtaga aaagggcgac 20 67 20 DNA Artificial Sequence AntisenseOligonucleotide 67 tggttctgat cagttccggc 20 68 20 DNA ArtificialSequence Antisense Oligonucleotide 68 cccatgatgg ttctgatcag 20 69 20 DNAArtificial Sequence Antisense Oligonucleotide 69 tgtccagccc atgatggttc20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 ctcccggaggaagtccaatg 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71gccgctcccg gaggaagtcc 20 72 20 DNA Artificial Sequence AntisenseOligonucleotide 72 gtcttggatc cagcccaaca 20 73 20 DNA ArtificialSequence Antisense Oligonucleotide 73 ccaccctggt cttggatcca 20 74 20 DNAArtificial Sequence Antisense Oligonucleotide 74 gtcccaacca ccctggtctt20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 aggaggccgtcccaaccacc 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76gtaggagagg aggccgtccc 20 77 20 DNA Artificial Sequence AntisenseOligonucleotide 77 agatggtcac ggtctgccac 20 78 20 DNA ArtificialSequence Antisense Oligonucleotide 78 aaagatggtc acggtctgcc 20 79 20 DNAArtificial Sequence Antisense Oligonucleotide 79 cgccacaaag atggtcacgg20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 ttccagatggtgagcgaggc 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81tcttccagat ggtgagcgag 20 82 20 DNA Artificial Sequence AntisenseOligonucleotide 82 cttcttccag atggtgagcg 20 83 20 DNA ArtificialSequence Antisense Oligonucleotide 83 catcttcttc cagatggtga 20 84 20 DNAArtificial Sequence Antisense Oligonucleotide 84 cagcccatct tcttccagat20 85 20 DNA Artificial Sequence Antisense Oligonucleotide 85 gcacagggccttgagcacca 20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86aatgcccatg tcccccaatc 20 87 20 DNA Artificial Sequence AntisenseOligonucleotide 87 ctcaagacca cttttcccca 20 88 20 DNA ArtificialSequence Antisense Oligonucleotide 88 tttttgggtc ccgaaggagg 20 89 20 DNAArtificial Sequence Antisense Oligonucleotide 89 cccaccttga gcaccagttt20 90 20 DNA Artificial Sequence Antisense Oligonucleotide 90 agctgcccaccttgagcacc 20 91 20 DNA Artificial Sequence Antisense Oligonucleotide 91tccagtaaat gcttgttgaa 20 92 20 DNA Artificial Sequence AntisenseOligonucleotide 92 tcacccctgc acgtgaactc 20 93 20 DNA ArtificialSequence Antisense Oligonucleotide 93 tgaaccaaga tcatgccatt 20 94 20 DNAArtificial Sequence Antisense Oligonucleotide 94 ggcagaggtt gcagtgaacc20 95 20 DNA Artificial Sequence Antisense Oligonucleotide 95 caaatccgtcttccaaataa 20 96 20 DNA Artificial Sequence Antisense Oligonucleotide 96aggttgcgcc attgcactcc 20 97 20 DNA Artificial Sequence AntisenseOligonucleotide 97 tggcacatgc ctgtaatccc 20 98 20 DNA ArtificialSequence Antisense Oligonucleotide 98 atacaaaatt accgggcgtg 20 99 20 DNAArtificial Sequence Antisense Oligonucleotide 99 cttggtgcac agggcctgtg20 100 20 DNA Artificial Sequence Antisense Oligonucleotide 100gatgaaaccc cgcctctggg 20 101 2707 DNA Mus musculus CDS (2674)...(2707)101 ctagattagg ttggcttgtt tgtgggaatt atctttgttg attgaagcgc caagtctcag 60cccactgtgg gtgactccat tccctaggca gggacttctg gactatgtag gagtggagaa 120agtaaactca gcattgacct ggatacattc attcaccaca ctttgctgtt gactatggat 180gtgatgtggc taggccctgc tggtgtgacg ttcctgctct ggctgtcctt gaactcagag 240agatggtcct gcatctccct cctgtgtgct aggtttcaaa ccatgcacca caatgcccag 300ctatgttgat tgattgattt tcatgtgaaa cacctctcaa ggcccaggga catatctgac 360ttctgagaag acttgagttt caaaggcagc cactgtctct agtacatcag ccaggcatta 420aaggatacat ttggagatca ccatgtgtca agcagtgtgc tagtttctag ggaattgaga 480acaaatgaat acagtcttct tgggaaacca aggtaggtga gagccaccag caatgaacga 540aaacactgca cacaaaatta attacagtgc tgtgaagcat aataaaaccc agtggttaaa 600aaaagagaaa agaaaagaaa agaaaagaag agaaaagaaa gccgggcgtg gtggcgcacg 660cctttaatcc cagcactggg gaggcagagg caggcggatt tcggagttcg aggccagcct 720ggtctacaaa gtgagttcca ggatagccag ggctatacag agaaaccctg tctcaaaaaa 780cccaccaaaa cagaaaagaa aagaaaagaa aagaaaagaa gggaaaagag aagaaaaagg 840agagctgccc gtggtggtgc acaccttcaa tccccgcaca gaagaagagg caggcggttc 900tctatgttgg aggccagcct ggtctacaga ctgagttcca ggacagccag ggctccacag 960aagctgtttc aaataacaaa caaaaactct gggtgctttc agagcttttt caacagagga 1020ctcaagccaa gaggctttct gacacccaga taactctacc cttcccctgc tcaaaacttc 1080ccctgggtct caggtctcag acaccctcag aagactgata aattcctgca agaaaactcc 1140agagtgtctg tcctggcctg ctgctacttc acattattcc tcctcttcct ccttcttctc 1200ttttttttct ttctcttctt tttttttttt ccaaaccagg gtttctctaa gtagccatgg 1260ctgtcctgga actggctttg tagtccaggc tgacctcgaa ctcagagatc tactttcctg 1320cgttctgagg gctgggatta aaagcataca caaccaccat ctgactcttt tttcttcttc 1380ctcctcttct ccttcctcct cctcctcttc cttttgcttg gcatcctagg ctggcttcag 1440attcacagca attcttcata ggttctggga ttacaggtat gggccaccat accagggtct 1500ctttgcaact cccatcttcc tcccggtttc ttgttttgat taaggatctc agtatctaac 1560ttaattgacc tgtcacttgc tatgtagact tgtgttttgt aaggtatgag tgaggtatgg 1620tgaggtgaga agggtacctc agagggccca tcactgagaa atcccttcct cctgaagtac 1680cggccatgta gtatgtagca ccagatgatg tagtatggaa tagagtttat ttaggacatg 1740ggaagtagag tttgggaaag gaggagagag agagaaagga ggggaggaga agtagtggag 1800agagaagaga ccctccagaa acacctggaa ggcggggaag gggagagaag gagaaacggg 1860gcagggagag aaggggcagg aagtaagaga ataagacaag taagtgagca ggagagcgag 1920gagggggcaa acagtcctta tatagtaggc taggcatacc tgactgttgc caggtaacta 1980tggggcggag cctagaagga ctgcttctag gatgtctcaa tttcacaggc atctgcttgc 2040ttgtagttcc caaggctgaa attaaagatg ttgtagccac cgcgtacagc ctgtttttgt 2100gttttgagat agattcttac ttaatggtgc agcttggact caaactattt tatccaggct 2160tgcctcgaac tctttagcaa tcttcttgct tcactctatc agaggtatgg accatcactc 2220tggtcaactg gttttttttt tttttctccc cagactggag caatctctta tagtgcaggt 2280tggttttgag ctcgaggcaa tactcccgtc ctacctcagc ctctcaatgc tgggatgaca 2340agatatccca ggcaagcttt gaacttgcgg caattctgct ttaacctcct tagtgctctc 2400taccatgaat ctatgggaag aagaaataat gggggcgggg ggggaaacaa ccaactctgg 2460gcatcagttc ggattaaggt cgatccgcgc atgcgttcat ttagtacccg cggccccgcc 2520cctgcagcga gcgatgatga tcacgtgact agtcctgcgg ggcggaggcc atgttgcggg 2580gcacccacgt gagggccgca cgtccacgat cagtcacgtg accgtggtgc gccgcagccg 2640ccggggcgca cccggcgaga ggcagcggca gtg atg gac ggg tcc ggg gag cag 2694Met Asp Gly Ser Gly Glu Gln 1 5 ctt ggg agc ggc g 2707 Leu Gly Ser Gly10 102 457 DNA Mus musculus 102 agaaggccag cgccacctcc tcccacccccagctggggtc gtgtttgctt ttggcattct 60 gctctctggg tttgctgtgg agctgggatgcaggccgggt cccgccccct gtccatcaga 120 agcagtagcc aggccttcca tgctacttgtcactactagg gtccccagct ctgtctcccc 180 tcagggttat gagcctacct atccatccccctgactctcc ctgggaccca ggagtccagg 240 cacccctttc ctcctctctc ccccagggcccaccagctct gaacagatca tgaagacagg 300 ggcctttttg ctacagggtt tcatccaggatcgagcaggg aggatggctg gggagacact 360 gagctgacct tggagcagcc gccccaggatgcgtccacca agaagctgag cgagtgtctc 420 cggcgaattg gagatgaact ggacagcaatatggagc 457 103 20 DNA Artificial Sequence Antisense Oligonucleotide 103cctgtcttca tgatctgttc 20 104 20 DNA Artificial Sequence AntisenseOligonucleotide 104 ggtgtctccc cagccatcct 20 105 20 DNA ArtificialSequence Antisense Oligonucleotide 105 cagctcaggt gtctccccag 20 106 20DNA Artificial Sequence Antisense Oligonucleotide 106 ccaaggtcagctcaggtgtc 20 107 20 DNA Artificial Sequence Antisense Oligonucleotide107 gctgctccaa ggtcagctca 20 108 20 DNA Artificial Sequence AntisenseOligonucleotide 108 gggcggctgc tccaaggtca 20 109 20 DNA ArtificialSequence Antisense Oligonucleotide 109 ccaattcgcc ggagacactc 20 110 20DNA Artificial Sequence Antisense Oligonucleotide 110 ctgcagctccatattgctat 20 111 20 DNA Artificial Sequence Antisense Oligonucleotide111 tcagcaatca tcctctgcag 20 112 20 DNA Artificial Sequence AntisenseOligonucleotide 112 ccacgtcagc aatcatcctc 20 113 20 DNA ArtificialSequence Antisense Oligonucleotide 113 tccgtgtcca cgtcagcaat 20 114 20DNA Artificial Sequence Antisense Oligonucleotide 114 ggagtccgtgtccacgtcag 20 115 20 DNA Artificial Sequence Antisense Oligonucleotide115 tgccacccgg aagaagacct 20 116 20 DNA Artificial Sequence AntisenseOligonucleotide 116 gcaaacatgt cagctgccac 20 117 20 DNA ArtificialSequence Antisense Oligonucleotide 117 ttgccatcag caaacatgtc 20 118 20DNA Artificial Sequence Antisense Oligonucleotide 118 agttgaagttgccatcagca 20 119 20 DNA Artificial Sequence Antisense Oligonucleotide119 cccagttgaa gttgccatca 20 120 20 DNA Artificial Sequence AntisenseOligonucleotide 120 ccacgcggcc ccagttgaag 20 121 20 DNA ArtificialSequence Antisense Oligonucleotide 121 agtttgctag caaagtagaa 20 122 20DNA Artificial Sequence Antisense Oligonucleotide 122 tgagcaccagtttgctagca 20 123 20 DNA Artificial Sequence Antisense Oligonucleotide123 acagggcctt gagcaccagt 20 124 20 DNA Artificial Sequence AntisenseOligonucleotide 124 tagtgcacag ggccttgagc 20 125 20 DNA ArtificialSequence Antisense Oligonucleotide 125 tttagtgcac agggccttga 20 126 20DNA Artificial Sequence Antisense Oligonucleotide 126 ctcgggcactttagtgcaca 20 127 20 DNA Artificial Sequence Antisense Oligonucleotide127 agctcgggca ctttagtgca 20 128 20 DNA Artificial Sequence AntisenseOligonucleotide 128 gatcagctcg ggcactttag 20 129 20 DNA ArtificialSequence Antisense Oligonucleotide 129 tggttctgat cagctcgggc 20 130 20DNA Artificial Sequence Antisense Oligonucleotide 130 ccatgatggttctgatcagc 20 131 20 DNA Artificial Sequence Antisense Oligonucleotide131 aagtccagtg tccagcccat 20 132 20 DNA Artificial Sequence AntisenseOligonucleotide 132 aggaagtcca gtgtccagcc 20 133 20 DNA ArtificialSequence Antisense Oligonucleotide 133 cggaggaagt ccagtgtcca 20 134 20DNA Artificial Sequence Antisense Oligonucleotide 134 gatccagacaagcagccgct 20 135 20 DNA Artificial Sequence Antisense Oligonucleotide135 tggtcttgga tccagacaag 20 136 20 DNA Artificial Sequence AntisenseOligonucleotide 136 tcccagccac cctggtcttg 20 137 20 DNA ArtificialSequence Antisense Oligonucleotide 137 atggtcactg tctgccatgt 20 138 20DNA Artificial Sequence Antisense Oligonucleotide 138 aagatggtcactgtctgcca 20 139 20 DNA Artificial Sequence Antisense Oligonucleotide139 gccacaaaga tggtcactgt 20 140 20 DNA Artificial Sequence AntisenseOligonucleotide 140 aggactccag ccacaaagat 20 141 20 DNA ArtificialSequence Antisense Oligonucleotide 141 cgaggcggtg aggactccag 20 142 20DNA Artificial Sequence Antisense Oligonucleotide 142 tccagatggtgagcgaggcg 20 143 20 DNA Artificial Sequence Antisense Oligonucleotide143 ttcttccaga tggtgagcga 20 144 20 DNA Artificial Sequence AntisenseOligonucleotide 144 tcagcccatc ttcttccaga 20 145 20 DNA ArtificialSequence Antisense Oligonucleotide 145 gcaggaccat ctctctgagt 20 146 20DNA Artificial Sequence Antisense Oligonucleotide 146 tggtttgaaacctagcacac 20 147 20 DNA Artificial Sequence Antisense Oligonucleotide147 agatatgtcc ctgggccttg 20 148 20 DNA Artificial Sequence AntisenseOligonucleotide 148 aaactagcac actgcttgac 20 149 20 DNA ArtificialSequence Antisense Oligonucleotide 149 cctaccttgg tttcccaaga 20 150 20DNA Artificial Sequence Antisense Oligonucleotide 150 gctgggattaaaggcgtgcg 20 151 20 DNA Artificial Sequence Antisense Oligonucleotide151 aaatccgcct gcctctgcct 20 152 20 DNA Artificial Sequence AntisenseOligonucleotide 152 ggcctccaac atagagaacc 20 153 20 DNA ArtificialSequence Antisense Oligonucleotide 153 atctgggtgt cagaaagcct 20 154 20DNA Artificial Sequence Antisense Oligonucleotide 154 cgcaggaaagtagatctctg 20 155 20 DNA Artificial Sequence Antisense Oligonucleotide155 agcaagtgac aggtcaatta 20 156 20 DNA Artificial Sequence AntisenseOligonucleotide 156 aaacaggctg tacgcggtgg 20 157 20 DNA ArtificialSequence Antisense Oligonucleotide 157 agttgaccag agtgatggtc 20 158 20DNA Artificial Sequence Antisense Oligonucleotide 158 gtcacgtgatcatcatcgct 20 159 20 DNA Artificial Sequence Antisense Oligonucleotide159 ggacccgtcc atcactgccg 20 160 20 DNA Artificial Sequence AntisenseOligonucleotide 160 actgcttctg atggacaggg 20 161 20 DNA ArtificialSequence Antisense Oligonucleotide 161 ggcctggcta ctgcttctga 20 162 20DNA Artificial Sequence Antisense Oligonucleotide 162 gcatggaaggcctggctact 20 163 20 DNA Artificial Sequence Antisense Oligonucleotide163 gtagtgacaa gtagcatgga 20 164 20 DNA Artificial Sequence AntisenseOligonucleotide 164 accctagtag tgacaagtag 20 165 20 DNA ArtificialSequence Antisense Oligonucleotide 165 taggctcata accctgaggg 20 166 20DNA Artificial Sequence Antisense Oligonucleotide 166 atggataggtaggctcataa 20 167 20 DNA Artificial Sequence Antisense Oligonucleotide167 ctggactcct gggtcccagg 20 168 20 DNA Artificial Sequence AntisenseOligonucleotide 168 tcagagctgg tgggccctgg 20

What is claimed is:
 1. A compound 8 to 50 nucleobases in length targetedto a nucleic acid molecule encoding BCL2-associated X protein, whereinsaid compound specifically hybridizes with said nucleic acid moleculeencoding BCL2-associated X protein and inhibits the expression ofBCL2-associated X protein.
 2. The compound of claim 1 which is anantisense oligonucleotide.
 3. The compound of claim 2 wherein theantisense oligonucleotide has a sequence comprising SEQ ID NO: 23, 24,25, 26, 28, 29, 30, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 47,48, 49, 50, 51, 52, 53, 54, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 89, 90, 91, 92, 94, 96, 97, 46, 103, 104, 105, 106, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 124, 125, 126, 127,129, 130, 131, 132, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,147, 150, 158, 159, 160, 162, 163, 164, 165, 166, 167 or
 168. 4. Thecompound of claim 2 wherein the antisense oligonucleotide comprises atleast one modified internucleoside linkage.
 5. The compound of claim 4wherein the modified internucleoside linkage is a phosphorothioatelinkage.
 6. The compound of claim 2 wherein the antisenseoligonucleotide comprises at least one modified sugar moiety.
 7. Thecompound of claim 6 wherein the modified sugar moiety is a2′-O-methoxyethyl sugar moiety.
 8. The compound of claim 2 wherein theantisense oligonucleotide comprises at least one modified nucleobase. 9.The compound of claim 8 wherein the modified nucleobase is a5-methylcytosine.
 10. The compound of claim 2 wherein the antisenseoligonucleotide is a chimeric oligonucleotide.
 11. A compound 8 to 50nucleobases in length which specifically hybridizes with at least an8-nucleobase portion of an active site on a nucleic acid moleculeencoding BCL2-associated X protein.
 12. A composition comprising thecompound of claim 1 and a pharmaceutically acceptable carrier ordiluent.
 13. The composition of claim 12 further comprising a colloidaldispersion system.
 14. The composition of claim 12 wherein the compoundis an antisense oligonucleotide.
 15. A method of inhibiting theexpression of BCL2-associated X protein in cells or tissues comprisingcontacting said cells or tissues with the compound of claim 1 so thatexpression of BCL2-associated X protein is inhibited.
 16. A method oftreating an animal having a disease or condition associated withBCL2-associated X protein comprising administering to said animal atherapeutically or prophylactically effective amount of the compound ofclaim 1 so that expression of BCL2-associated X protein is inhibited.17. The method of claim 16 wherein the disease or condition arises fromaberrant apoptosis.
 18. The method of claim 16 wherein the disease orcondition is familial amyotrophic lateral sclerosis, Alzeheimer'sdisease, Parkinson's disease, Hodgkin's disease, cartilage-hairhyperplasia, diabetes-associated ocular disorders or scrapie infections.19. The compound of claim 1 targeted to a nucleic acid molecule encodingBCL2-associated X protein, wherein said compound specifically hybridizeswith and inhibits the expression of an alternatively spliced variant ofBCL2-associated X protein.
 20. The compound of claim 19 wherein saidalternatively spliced variant is BAX-alpha, BAX-beta, BAX-gamma,BAX-delta, BAX-omega or BAX-epsilon.